input to the “Particle subtraction” job-type. This new
subtraction utility will also allow to change the center and the
box size of the subtracted particles.
23. Details about performing conventional and focused classifications are provided in the RELION tutorial, which is available
from the RELION Wiki. General advice on classification strategies is also provided in [14].
Acknowledgments
We are grateful to Clemens Plaschka for discussions; to Jake Grimmett and Toby Darling for help with high-performance computing;
and to Shabih Shakeel, Rafael Fernandez-Leiro, and Giulia Zanetti
for critical reading of the manuscript. This study was supported by
the MRC-LMB EM facility. Funding was provided by the UK
Medical Research Council to SHWS (MC_UP_A025_1013); and
the Japan Society for the Promotion of Science through an Overseas Research Fellowship to TN.
References
1. Fernandez-Leiro R, Scheres SHW (2016)
Unravelling biological macromolecules with
cryo-electron
microscopy.
Nature
537:339–346.
https://doi.org/10.1038/
nature19948
2. Nakane T, Kimanius D, Lindahl E, Scheres SH
(2018) Characterisation of molecular motions
in cryo-EM single-particle data by multi-body
refinement in RELION. elife 7:e36861.
https://doi.org/10.7554/eLife.36861
3. Bai X, Rajendra E, Yang G et al (2015) Sampling the conformational space of the catalytic
subunit of human gamma-secretase. elife 4:
e11182.
https://doi.org/10.7554/eLife.
11182
4. Zhou Q, Huang X, Sun S et al (2015) CryoEM structure of SNAP-SNARE assembly in
20S particle. Cell Res 25:551–560. https://
doi.org/10.1038/cr.2015.47
5. Ilca SL, Kotecha A, Sun X et al (2015) Localized reconstruction of subunits from electron
cryomicroscopy images of macromolecular
complexes. Nat Commun 6:8843. https://
doi.org/10.1038/ncomms9843
6. Plaschka C, Lin P-C, Nagai K (2017) Structure
of a pre-catalytic spliceosome. Nature
546:617–621.
https://doi.org/10.1038/
nature22799
7. Iudin A, Korir PK, Salavert-Torres J et al
(2016) EMPIAR: a public archive for raw
electron microscopy image data. Nat Methods
13:387–388.
https://doi.org/10.1038/
nmeth.3806
8. Zivanov J, Nakane T, Forsberg BO et al (2018)
New tools for automated high-resolution cryoEM structure determination in RELION-3.
elife 7:e42166. https://doi.org/10.7554/
eLife.42166
9. Pettersen EF, Goddard TD, Huang CC et al
(2004) UCSF chimera--a visualization system
for exploratory research and analysis. J Comput
Chem 25:1605–1612. https://doi.org/10.
1002/jcc.20084
10. Emsley P, Lohkamp B, Scott WG, Cowtan K
(2010) Features and development of Coot.
Acta Crystallogr D Biol Crystallogr
66:486–501.
https://doi.org/10.1107/
S0907444910007493
11. Pintilie G, Chiu W (2012) Comparison of Segger and other methods for segmentation and
rigid-body docking of molecular components
in cryo-EM density maps. Biopolymers
97:742–760. https://doi.org/10.1002/bip.
22074
12. Chen S, McMullan G, Faruqi AR et al (2013)
High-resolution noise substitution to measure
overfitting and validate resolution in 3D structure determination by single particle electron
cryomicroscopy. Ultramicroscopy 135:24–35.
Multi-body Refinement in RELION
159
subtraction utility will also allow to change the center and the
box size of the subtracted particles.
23. Details about performing conventional and focused classifications are provided in the RELION tutorial, which is available
from the RELION Wiki. General advice on classification strategies is also provided in [14].
Acknowledgments
We are grateful to Clemens Plaschka for discussions; to Jake Grimmett and Toby Darling for help with high-performance computing;
and to Shabih Shakeel, Rafael Fernandez-Leiro, and Giulia Zanetti
for critical reading of the manuscript. This study was supported by
the MRC-LMB EM facility. Funding was provided by the UK
Medical Research Council to SHWS (MC_UP_A025_1013); and
the Japan Society for the Promotion of Science through an Overseas Research Fellowship to TN.
References
1. Fernandez-Leiro R, Scheres SHW (2016)
Unravelling biological macromolecules with
cryo-electron
microscopy.
Nature
537:339–346.
https://doi.org/10.1038/
nature19948
2. Nakane T, Kimanius D, Lindahl E, Scheres SH
(2018) Characterisation of molecular motions
in cryo-EM single-particle data by multi-body
refinement in RELION. elife 7:e36861.
https://doi.org/10.7554/eLife.36861
3. Bai X, Rajendra E, Yang G et al (2015) Sampling the conformational space of the catalytic
subunit of human gamma-secretase. elife 4:
e11182.
https://doi.org/10.7554/eLife.
11182
4. Zhou Q, Huang X, Sun S et al (2015) CryoEM structure of SNAP-SNARE assembly in
20S particle. Cell Res 25:551–560. https://
doi.org/10.1038/cr.2015.47
5. Ilca SL, Kotecha A, Sun X et al (2015) Localized reconstruction of subunits from electron
cryomicroscopy images of macromolecular
complexes. Nat Commun 6:8843. https://
doi.org/10.1038/ncomms9843
6. Plaschka C, Lin P-C, Nagai K (2017) Structure
of a pre-catalytic spliceosome. Nature
546:617–621.
https://doi.org/10.1038/
nature22799
7. Iudin A, Korir PK, Salavert-Torres J et al
(2016) EMPIAR: a public archive for raw
electron microscopy image data. Nat Methods
13:387–388.
https://doi.org/10.1038/
nmeth.3806
8. Zivanov J, Nakane T, Forsberg BO et al (2018)
New tools for automated high-resolution cryoEM structure determination in RELION-3.
elife 7:e42166. https://doi.org/10.7554/
eLife.42166
9. Pettersen EF, Goddard TD, Huang CC et al
(2004) UCSF chimera--a visualization system
for exploratory research and analysis. J Comput
Chem 25:1605–1612. https://doi.org/10.
1002/jcc.20084
10. Emsley P, Lohkamp B, Scott WG, Cowtan K
(2010) Features and development of Coot.
Acta Crystallogr D Biol Crystallogr
66:486–501.
https://doi.org/10.1107/
S0907444910007493
11. Pintilie G, Chiu W (2012) Comparison of Segger and other methods for segmentation and
rigid-body docking of molecular components
in cryo-EM density maps. Biopolymers
97:742–760. https://doi.org/10.1002/bip.
22074
12. Chen S, McMullan G, Faruqi AR et al (2013)
High-resolution noise substitution to measure
overfitting and validate resolution in 3D structure determination by single particle electron
cryomicroscopy. Ultramicroscopy 135:24–35.
Multi-body Refinement in RELION
159